Fusion protein containing improved anti-VEGFR2 (KDR) antibody and its uses

Abstract

The present invention relates to a fusion protein containing an improved anti-VEGFR2 (KDR) antibody or an antigen-binding fragment thereof with improved properties and its uses. Specifically, the present invention relates to a fusion protein in which a DLL4 (Delta-like ligand 4) binding protein domain is bound to the end of an anti-VEGFR2 (KDR) antibody in which the heavy chain FR (framework regoin) of a human antibody that specifically binds to VEGFR2 / KDR is replaced from VH1 to VH3 and the light chain FR (framework regoin) is replaced from VL1 to VK1, and the affinity to the antigen is increased by inducing mutations, a composition for diagnosing or treating angiogenesis-related diseases, a composition for preventing or treating tumors or cancers, and a composition for co-administration with a therapeutic agent other than the fusion protein, which contains the fusion protein.

Description

[Technical Field]

[0001] The present invention relates to a fusion protein comprising an improved anti-VEGFR2 (KDR) antibody or its antigen-binding fragment exhibiting improved properties, and uses thereof. Specifically, the present invention relates to a fusion protein in which a DLL4 (Delta-like ligand 4) binding protein domain is attached to the terminus of an anti-VEGFR2 (KDR) antibody that has been mutated by substituting the heavy chain framework FR (FR) from VH1 to VH3 and the light chain framework FR (FR) from VL1 to VK1 of a human antibody that specifically binds to VEGFR2 / KDR to increase its affinity for the antigen; compositions containing the fusion protein for diagnosing or treating diseases associated with angiogenesis, compositions for preventing or treating tumors or cancer, and compositions for co-administering with therapeutic agents other than the fusion protein.

[0002] [Background technology]

[0003] VEGFR2 / KDR is the primary angiogenic receptor, binding to VEGF isoforms A, C, D, and E, and is important for the mitogenic, angiogenic, and permeability-enhancing effects of VEGF as well as endothelial cell differentiation. Anti-VEGFR2 antibodies can prevent all known VEGF isoforms from binding to VEGFR2 / KDR and initiating signaling. Furthermore, because tumors secrete higher amounts of VEGF while the number of receptors remains relatively constant, targeting the receptor increases the likelihood of completely inhibiting signaling, even in the presence of very high levels of VEGF isoforms.

[0004] Angiogenesis refers to the generation of new blood vessels from existing ones through the growth, division, and migration of endothelial cells. Angiogenesis does not occur in adults except for special circumstances such as wound healing and the female reproductive cycle. However, excessive angiogenesis has been reported in diseases such as tumor growth and metastasis, age-related macular denaturation, rheumatoid arthritis, diabetic retinopathy, psoriasis, and chronic inflammation (Cameliet and Jain, Nature, 407:249, 2000). For this reason, many efforts are being made to treat diseases, especially tumors, using angiogenesis inhibitors.

[0005] In 1971, Dr. J. Folkman proposed that tumor growth and metastasis are dependent on angiogenesis, and therefore that anti-angiogenesis-focused therapy could be a new therapeutic agent for solid cancers. Since then, research into inhibiting excessive angiogenesis has attracted the attention of many researchers (Ferrara and Kerbel, Nature, 435:967, 2005). The progression of the angiogenesis process is determined by the overall balance of angiogenesis-inducing factors and angiogenesis-inhibiting factors, and progresses through a complex, sequential process consisting of multiple steps. The process begins with tumors and injured tissues secreting various angiogenesis-inducing factors, including vascular endothelial growth factor (VEGF), which binds to the appropriate receptors on surrounding existing vascular endothelial cells, activating them and increasing their permeability. They then secrete proteolytic enzymes, such as matrix metalloproteinases (MMPs), which degrade the basement membrane and extracellular matrix around the endothelial cells. This causes the endothelial cells to migrate and proliferate away from existing capillaries toward the tissue secreting the angiogenesis-inducing factors. The migrated and proliferated endothelial cells then form intravascular tube structures. Finally, with the influx of pericytes, which provide structural support for the endothelial cells, stable and mature blood vessels are formed.

[0006] DLL4 is one of the key factors responsible for normal vascular tube formation during the angiogenesis process. DLL4 is specifically induced by VEGF and acts on Notch receptors to appropriately regulate angiogenic sprouting. Blockade of DLL4 results in a loss of lateral inhibition in cells at the end of angiogenesis sites, leading to excessive sprouting. This results in excessively abundant but less productive angiogenesis, which in turn impairs oxygen perfusion and induces hypoxia around the tumor. In experimental cancer models, activation of DLL4 signaling enhanced angiogenesis, while inhibition inhibited tumor growth (Lobov IB et al., Proc Natl Acad Sci U S A. 2007 Feb 27;104(9):3219-24).

[0007] DLL4 is one of the ligands for the Notch receptor, and it is known that there are currently four types of Notch receptors (Notch 1 to 4) and five types of Notch ligands (Jagged-1, Jagged-2, DLL1, DLL3, and DLL4) in mammals. The Notch signaling system begins when a Notch ligand on one cell binds to a Notch receptor on another cell, but it can only be activated by direct interaction between different cells (Bray SJ, Nat Rev Mol Cell Biol., 7(9):678, 2006).

[0008] When a Notch ligand binds to the Notch receptor, ADAM metalloproteases are activated, cleaving the proximal region on the outer side of the Notch receptor membrane. Subsequently, the gamma-secretase complex is activated, cleaving the proximal region on the inner side of the Notch receptor membrane, releasing the Notch intracellular domain (NICD). NICD then translocates to the nucleus and binds to RBPJ / CSL transcription factors, inducing the expression of Notch target genes, such as basic helix-loop-helix proteins like Hes and Hey. Thus, the Notch signaling pathway determines the fate of a cell—proliferation, differentiation, or apoptosis—depending on the cell's current conditions, and also plays an important role in maintaining both normal and cancer stem cells.

[0009] Although all Notch receptors can bind to all Notch ligands, the combination of these binding events is selectively regulated by the microenvironment in which the cells reside. For example, DLL4 is strongly expressed in endothelial cells during angiogenesis during fetal development and binds to Notch1 and Notch4, both of which are expressed in surrounding endothelial cells. Of these, DLL4-Notch1 binding is exclusively the most important factor in promoting angiogenesis (Yan M, Vasc Cell, 2011). This binding is essential for promoting angiogenesis. This fact has been well documented through gene depletion experiments (Duarte et al., Genes Dev, 2004; Gale et al., PNAS, 2004; Krebs et al., Genes Dev, 2004).

[0010] Therefore, inhibiting DLL4-Notch1 binding suppresses angiogenesis, which can be used to treat various diseases, including tumors. It has already been demonstrated that suppressing VEGF with drugs such as Avastin (bevacizumab) in cancer treatment suppresses angiogenesis, resulting in reduced tumor perfusion and a decrease in tumor size. On the other hand, targeting DLL4 and inhibiting its binding to Notch1 expressed in surrounding cells results in hypersprouting but non-functional blood vessels, reducing tumor perfusion and ultimately reducing tumor size (Thurston et al., Nat Rev Cancer, 7(5):327, 2007).

[0011] Therefore, the present researchers attempted to effectively inhibit neovascularization by creating a dual-targeting antibody in which Notch1, particularly the DLL4-binding site of Notch1 in the present invention, was fused to an existing antibody.

[0012] Factors involved in angiogenesis include vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor (TGFb), and fibroblast growth factor (FGF). Among these, endothelial growth factor (VEGF) is an endothelial cell-specific factor that is directly involved in the growth, differentiation, and migration of endothelial cells. There are four distinct isoforms (VEGF165, VEGF121, VEGF189, and VEGF206), and VEGF165 is the most abundant isoform in all human tissues except the placenta (Tisher et al., J Biol Chem, 266:11947, 1991).

[0013] Endothelial growth factor (VEGF) regulates the formation of new blood vessels in situ from the differentiation of endothelial precursors (vascular progenitors) and is expressed in embryonic tissues (Breier et al., Development (Camb), 114:521, 1992), macrophages, and proliferating epidermal keratinocytes during wound healing (Brown et al., J. Exp. Med., 176:1375, 1992), and may be responsible for tissue edema associated with inflammation (Ferrara et al., Endocr. Rev., 13:18, 1992). In situ hybridization studies have demonstrated that VEGF is highly expressed in numerous human tumor lines, including glioblastoma multiforme, hemangioblastoma, central nervous system neoplasms, and AIDS-associated Kaposi's sarcoma (Plate et al., Nature 359:845, 1992; Plate et al., Cancer Res. 53:5822, 1993; Berkman et al., J. Clin. Invest. 91:153, 1993; Nakamura et al., AIDS Weekly. 13(1), 1992). High levels of VEGF have also been observed in hypoxia-induced angiogenesis (Shweiki et al., Nature 359:843, 1992).

[0014] The biological functions of VEGF are mediated by VEGF receptors, which have high affinity for VEGF and are selectively expressed in endothelial cells during embryogenesis (Millauer et al., Cell, 72:835, 1993) and tumorigenesis. VEGF receptors (VEGFRs) are generally class III receptor tyrosine kinases characterized by multiple, typically five or seven, immunoglobulin-like loops in their amino-terminal extracellular receptor ligand-binding domain (Kaipainen et al., J. Exp. Med., 178:2027, 1993). The other two domains contain a carboxy-terminal intracellular catalytic domain and a transmembrane domain interrupted by the insertion of a hydrophilic interkinase sequence of variable length, termed the kinase insert domain (Terman et al., Oncogene, 6:1677, 1991). VEGFRs include the fms-like tyrosine kinase receptor (flt-1), or VEGFR-1 (Shibuya et al., Oncogene, 5:519, 1990; WO92 / 14248; Terman et al., Oncogene, 6:1677, 1991) and the kinase insert domain receptor / fetal kinase (KDR / flk-1), or VEGFR-2 (Matthews et al., Proc. Natl. Acad. Sci. USA, 88:9026, 1991), although other receptors, such as neuropilin-1 and neuropilin-2, can also bind VEGF. Another tyrosine kinase receptor, VEGFR-3 (flt-4), binds to the VEGF homologs VEGF-C and VEGF-D and plays an important role in lymphatic vessel development.

[0015] High levels of Flk-1 are expressed by endothelial cells infiltrating glioblastomas (Plate et al., Nature 359:845, 1992). Flk-1 levels are specifically upregulated by VEGF produced by human glioblastomas (Plate et al., Cancer Res. 53:5822, 1993). The high expression of Flk-1 in glioblastoma terminal endothelial cells (GAECs) indicates that Flk-1 transcripts are barely detectable in normal brain endothelial cells, and that receptor activity is likely induced during tumorigenesis. Such upregulation occurs only in vascular endothelial cells in close proximity to the tumor. Blockade of VEGF activity with a neutralizing anti-VEGF monoclonal antibody (mAb) suppresses the growth of human tumor xenografts in nude mice (Kim et al., Nature 362:841, 1993), indicating a direct role for VEGF in tumor-associated angiogenesis.

[0016] VEGF ligands are upregulated in tumor cells, and their receptors are upregulated in tumor-infiltrating vascular endothelial cells, but expression of VEGF ligands and their receptors is low in normal cells not involved in angiogenesis. Therefore, these normal cells block the interaction between VEGF and its receptor, suppressing angiogenesis and thus preventing tumor growth.

[0017] High levels of VEGFR-2 are expressed by endothelial cells infiltrating gliomas and are specifically upregulated by VEGF produced by human glioblastomas (Plate et al., Nature, 359:845, 1992; Plate et al., Cancer Res., 53:5822, 1993). Because VEGFR-2 transcripts are barely detectable in normal brain endothelial cells, the high expression of VEGFR-2 in glioblastoma-associated endothelial cells (GAECs) indicates that receptor activity is induced during tumorigenesis.

[0018] VEGF is expressed at high levels in a variety of tumors (Plate et al., Nature, 359:8435, 1992; Dvorak et al., J. Exp. Med., 174:1275, 1991), and newly formed capillaries converge around VEGF-producing tumor alveoli (Plate et al., Nature, 359:8435, 1992). VEGF expression is strongly upregulated under hypoxic conditions, such as those associated with rapidly growing tumors (Shweiki et al., Nature, 359:843, 1992). VEGF neutralization with antibodies such as Avastin (Ferrara et al., Nat Rev Drug Discov., 3(5):391, 2004; Avery et al., Ophthalmology, 113(3):363, 2006) is a clinically approved therapy for inhibiting cancer or other angiogenic diseases such as AMD. However, resistance to VEGF blockade has been discovered, even when combined with chemotherapy. This resistance may be associated with a remodeled vasculature and increased expression of other angiogenic factors. Thus, there remains a need in the art for improved compositions and methods for inhibiting cancer and other diseases associated with angiogenesis.

[0019] Although both tyrosine kinase inhibitors (TKIs) and anti-KDR antibodies can inhibit KDR-mediated angiogenesis, antibody approaches have advantages over TKIs. In contrast to TKIs, anti-KDR antibodies are more specific KDR-targeting agents (i.e., they do not inhibit other VEGF receptors). Due to this high specificity, anti-KDR antibodies can limit and / or avoid off-target effects and toxicity caused by less specific TKIs (Witte et al., Cancer Metastasis Rev., Jun;17(2):155, 1998).

[0020] Unlike Avastin, which binds only one ligand (VEGF-A), anti-KDR antibodies are expected to prevent all known VEGFs from binding to VEGFR2 / KDR. Anti-KDR antibodies may have a more potent inhibitory effect on tumor angiogenesis than simply blocking VEGF-A. Anti-KDR antibody therapy may be effective in Avastin-resistant tumors.

[0021] Under these technical backgrounds, the inventors of the present application have produced a novel antibody with increased affinity for the antigen by substituting the framework region FR (framework region) of the heavy chain variable region from VH1 to VH3 and the framework region FR (framework region) of the light chain from VL1 to VK1 in order to improve an existing antibody that specifically binds to VEGFR2 / KDR. They also induced mutations in the CDR3 regions of the heavy and light chains, thereby producing a fusion protein by fusing the DLL4 binding site of Notch1 to the end of the antibody, thereby completing the present invention.

[0022]

[0023] Summary of the Invention [Problem to be solved by the invention]

[0024] An object of the present invention is to provide a fusion protein in the form of an antibody having improved binding ability to VEGFR2 / KDR fused with a DLL4-binding protein domain.

[0025] Another object of the present invention is to provide a nucleic acid encoding the fusion protein.

[0026] Another object of the present invention is to provide a vector containing the nucleic acid, a transformed cell containing the vector, and a method for producing the same.

[0027] It is still another object of the present invention to provide a composition for preventing or treating angiogenic diseases, comprising the fusion protein, a method for preventing or treating angiogenic diseases, comprising administering the fusion protein to an individual, and a use of the fusion protein for the preparation of a composition for preventing or treating angiogenic diseases.

[0028] It is still another object of the present invention to provide a composition for diagnosing angiogenic diseases, which comprises the fusion protein. It is also an object of the present invention to provide a method for diagnosing angiogenic diseases, which comprises administering the fusion protein to an individual or treating a sample with the fusion protein. It is also an object of the present invention to provide a use of the fusion protein for the preparation of a composition for diagnosing angiogenic diseases.

[0029] It is still another object of the present invention to provide a composition for preventing or treating tumors or cancer, which comprises the fusion protein. It is also an object of the present invention to provide a method for preventing or treating tumors or cancer, which comprises administering the fusion protein to an individual. It is also an object of the present invention to provide a use of the fusion protein for the manufacture of a composition for preventing or treating tumors or cancer.

[0030] It is still another object of the present invention to provide a composition for co-administration with other therapeutic agents, which comprises the fusion protein, a method for co-administration with other therapeutic agents, which comprises administering the fusion protein to an individual, and a use of the fusion protein for the preparation of a composition for co-administration with other therapeutic agents. [Means for solving the problem]

[0031] In order to achieve the above object, the present invention provides a fusion protein comprising an antibody or an antigen-binding fragment thereof that binds to VEGFR2 / KDR; and a DLL4 (Delta-like ligand 4) binding protein domain,

[0032] The antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR provides a fusion protein comprising a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, and a heavy chain CDR3 of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6.

[0033] The present invention also provides a nucleic acid encoding the fusion protein.

[0034] The present invention also provides a vector comprising the nucleic acid.

[0035] The present invention also provides a transformed cell containing the vector.

[0036] The present invention also provides a method for producing the fusion protein, comprising the steps of: (a) culturing the cells to produce the fusion protein; and (b) recovering the produced fusion protein.

[0037] The present invention also provides a composition for preventing or treating angiogenic diseases, comprising the fusion protein.

[0038] The present invention also provides a composition for diagnosing angiogenic diseases, which comprises the fusion protein.

[0039] The present invention also provides a composition for preventing or treating tumors or cancers, comprising the fusion protein.

[0040] The present invention also provides a composition comprising the fusion protein for co-administration with other therapeutic agents.

[0041] [Brief explanation of the drawings]

[0042] [Figure 1] This sequence is a grafted version of the heavy chain CDR sequence of 6A6.

[0043] [Figure 2] This sequence is a graft of the light chain CDR sequence of 6A6.

[0044] [Figure 3A-3B] The binding strength of the fusion protein was confirmed by SPR.

[0045] [Figure 4] The ability to simultaneously bind to two types of antigens was confirmed by SPR.

[0046] [Figure 5] This result confirmed that growth regulators can suppress the proliferation of HUVEC cells.

[0047] [Figure 6] These results confirm the inhibitory effect of antibodies on VEGFR-2 phosphorylation in vascular endothelial cells.

[0048] [Figure 7] The results show that the binding ability of the fusion protein in vascular endothelial cells was confirmed using FACS.

[0049] [Figure 8] The binding ability of the fusion protein to hDLL4 expressed on the cell surface was confirmed using FACS.

[0050]

[0051] DETAILED DESCRIPTION OF THE INVENTION

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein is well known and commonly used in the art.

[0053] The present invention relates to an antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR; and a fusion protein comprising a DLL4-binding protein domain, wherein the antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR comprises a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, and a heavy chain CDR3 of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6.

[0054] In the fusion protein of the present invention, the antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR is an improved version of a fully humanized antibody that neutralizes vascular endothelial growth factor receptor, particularly an improved version of a human monoclonal antibody that neutralizes vascular endothelial growth factor receptor (Korean Patent Registration No. 10-0883430).

[0055] The inventors of this application have developed a unique antibody, created from a fully human antibody library, that targets VEGFR-2 / KDR while simultaneously reacting with mouse or rat flk-1 (a VEGFR-2 homolog). Clinical trials have demonstrated the superiority of this antibody, but the antibody protein's binding affinity to the antigen is somewhat low and its in vivo half-life is short. By improving this antibody, its efficacy as an immunotherapy for cancer and angiogenesis-related diseases is expected to be enhanced.

[0056] To improve an existing antibody that specifically binds to VEGFR2 / KDR, the heavy chain variable region FR (framework region) was substituted from VH1 to VH3, and the light chain FR (framework region) was substituted from VL1 to VK1, and mutations were introduced into the CDR3 regions of the heavy and light chains to produce a novel antibody with increased affinity for the antigen. As a result, the inventors developed an antibody with higher binding affinity and better intracellular efficacy than existing antibodies that specifically bind to VEGFR2 / KDR, and confirmed that the antibody can function as a targeted immunotherapy for cancer or a therapeutic agent for angiogenesis-related diseases.

[0057] The improved antibodies were screened as follows: First, the CDR and framework (FR) sequences of 6A6 were confirmed, and then the heavy chain CDR sequence was grafted onto a human germline VH3 sequence, and the light chain CDR sequence was grafted onto a germline VK1 sequence using a CDR grafting technique.

[0058] CDR grafting generally involves isolating DNA encoding the variable region sequences of a mouse monoclonal antibody, obtaining the CDR sequences by gene cloning, and then grafting the CDR sequences into an appropriate human antibody sequence in silico (Hou S, et al. Humanization of an anti-CD34 monoclonal antibody by complementarity-determining region grafting based on computer-assisted molecular modeling. J. Biochem. 2008;144(1):115-20; and Kashmiri SV, De Pascalis R, Gonzales NR, Schlom J. SDR grafting—a new approach to antibody humanization. Methods. 2005;36(1):25-34). Using the above method, the 6A6 framework, which has a germline VH1 / VL1 region, was replaced with a germline VH3 / VK1 region.

[0059] To improve the affinity of the substituted antibodies, we constructed a library of mutations in the CDR3 base sequences of the light and heavy chains, and screened the antibodies using recombinant KDR D1-D3-Fc fusion proteins using phage display.

[0060] Based on this, the present invention includes an antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR, which comprises a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, and a heavy chain CDR3 of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6.

[0061] The fusion proteins of the invention may specifically bind to both VEGFR2 / KDR and DLL4. "Bispecific" or "dual specific" refers to a property of a binding protein that can specifically bind to two different targets and modulate the activity of the targets, and may be produced, for example, by an antibody or protein, or fragment thereof, that specifically binds to each target, and possesses two distinct antigen-binding arms (arms: specific for two targets) that are monovalent for each antigen that binds to it.

[0062] As used herein, the term "antibody" refers to an anti-VEGFR2 / KDR antibody that specifically binds to VEGFR2 / KDR. The scope of the present invention includes intact antibody forms that specifically bind to VEGFR2 / KDR, as well as antigen-binding fragments of such antibody molecules.

[0063] Intact antibodies have two full-length light chains and two full-length heavy chains, with each light chain connected to a heavy chain by a disulfide bond. Heavy chain constant regions are classified into gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, with subclasses of gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). Light chain constant regions are classified into kappa (κ) and lambda (λ) types.

[0064] An antigen-binding fragment of an antibody or antibody fragment refers to a fragment that retains antigen-binding function, and includes Fab, F(ab'), F(ab')2, and Fv. Among antibody fragments, Fab has a structure comprising light and heavy chain variable regions, a light chain constant region, and the first heavy chain constant region (CH1), and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are produced when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds. Fv is the smallest antibody fragment that comprises only the heavy chain variable region and the light chain variable region. In a two-chain Fv, the heavy chain variable region and the light chain variable region are linked non-covalently, while in a single-chain Fv (scFv), the heavy chain variable region and the light chain variable region are generally linked covalently via a peptide linker or directly at the C-terminus, and can form a dimer-like structure like the two-chain Fv. Such antibody fragments can be obtained using protease hydrolases (for example, Fab fragments can be obtained by limiting cleavage of a whole antibody with papain, or F(ab') fragments can be obtained by cleavage with pepsin), or can be produced by genetic recombination techniques.

[0065] In one embodiment, the antibodies of the present invention are in the form of Fv (e.g., scFv) or intact antibodies. The heavy chain constant region may be selected from any one of the gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) isotypes. For example, the constant region may be gamma 1 (IgG1), gamma 3 (IgG3), or gamma 4 (IgG4). The light chain constant region may be kappa or lambda.

[0066] As used herein, the term "heavy chain" refers to a full-length heavy chain or a fragment thereof, which includes a variable region domain VH containing an amino acid sequence with sufficient variable region sequence to confer antigen specificity, and three constant region domains CH1, CH2, and CH3. As used herein, the term "light chain" refers to a full-length light chain or a fragment thereof, which includes a variable region domain VL containing an amino acid sequence with sufficient variable region sequence to confer antigen specificity, and a constant region domain CL.

[0067] Antibodies of the present invention include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of these antibodies.

[0068] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0069] "Epitope" refers to a protein determinant to which an antibody can specifically bind. Epitopes are usually composed of chemically active surface groupings of molecules such as amino acids or sugar side chains and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0070] The "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate possessing the desired specificity, affinity, and capacity.

[0071] The term "human antibody" refers to a molecule derived from human immunoglobulin, in which the entire amino acid sequence constituting the antibody, including the complementarity determining regions and structural regions, is composed of human immunoglobulin.

[0072] Also included are "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remaining chains are identical to or homologous to corresponding sequences in antibodies from yet another species or belonging to yet another antibody class or subclass, as well as fragments of such antibodies that exhibit the desired biological activity.

[0073] As used herein, "antibody variable region" refers to the light and heavy chain portions of an antibody molecule that contain the amino acid sequences of the complementarity-determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs). VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0074] "Complementarity determining region (CDR; i.e., CDR1, CDR2, and CDR3)" refers to the amino acid residues of an antibody variable region that are necessary for antigen binding. Each variable region typically has three CDR regions identified as CDR1, CDR2, and CDR3. The present invention includes a heavy chain variable region comprising a heavy chain CDR3 of SEQ ID NO:3 and a light chain variable region comprising a light chain CDR3 of SEQ ID NO:6.

[0075] In the present invention, the antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR and DLL4 may comprise a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, and a heavy chain CDR3 of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6.

[0076] "Framework regions (FR)" are those variable region residues other than the CDR residues. Each variable region typically has four FRs, identified as FR1, FR2, FR3 and FR4.

[0077] An "Fv" fragment is an antibody fragment that contains a complete antigen recognition and binding site. Such region consists of a dimer of one heavy- and one light-chain variable region in tight, essentially covalent association, e.g., scFv.

[0078] A "Fab" fragment contains the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. F(ab')2 antibody fragments typically contain a pair of Fab fragments covalently linked near their carboxy termini by hinge cysteines between them.

[0079] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, but these domains are present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0080] VEGFR2 / KDR antibodies may comprise single chains or double chains. Functionally, the binding affinity of VEGFR2 / KDR antibodies is 10 -5 M~10 -12 For example, the binding affinity of VEGFR2 / KDR antibodies is in the range of 10 -6 M~10 -12 M, 10 -7 M~10 -12 M, 10 -8 M~10 -12 M, 10-9 M~10 -12 M, 10 -5 M~10 -11 M, 10 -6 M~10 -11 M, 10 -7 M~10 -11 M, 10 -8 M~10 -11 M, 10 -9 M~10 -11 M, 10 -10 M~10 -11 M, 10 -5 M~10 -10 M, 10 -6 M~10 -10 M, 10 -7 M~10 -10 M, 10 -8 M~10 -10 M, 10 -9 M~10 -10 M, 10 -5 M~10 -9 M, 10 -6 M~10 -9 M, 10 -7 M~10 -9 M, 10 -8 M~10 -9 M, 10 -5 M~10 -8 M, 10 -6 M~10 -8 M, 10 -7 M~10 -8 M, 10 -5 M~10 -7 M, 10 -6 M~10 -7 M or 10 -5 M~10 -6 I am M.

[0081] The present invention includes an antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR, which comprises a heavy chain variable region comprising a sequence having 80% or more homology to the sequence of SEQ ID NO:8.

[0082] The present invention also includes an antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR, which comprises a light chain variable region comprising a sequence having 80% or more homology to the sequence of SEQ ID NO:10.

[0083] The present invention also includes an antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR, comprising a heavy chain variable region comprising a sequence having 80% or more homology to the sequence of SEQ ID NO: 8 and a light chain variable region comprising a sequence having 80% or more homology to the sequence of SEQ ID NO: 10.

[0084] The homology may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology compared to the entire sequence of the claimed SEQ ID NO.

[0085] The present invention encompasses not only the fusion protein sequences described herein, but also their biological equivalents. For example, additional modifications can be made to the amino acid sequence to further improve the binding affinity and / or other biological properties of the fusion protein. Such modifications include, for example, deletion, insertion, and / or substitution of amino acid sequence residues. Such amino acid mutations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, and size. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine are considered to be biologically functional equivalents.

[0086] Considering the above-mentioned biologically equivalent mutations, the fusion protein of the present invention or the nucleic acid molecule encoding it is also understood to include sequences showing substantial identity to the sequences set forth in SEQ ID NO: 1. The term "substantial identity" refers to a sequence that shows at least 90% homology, most preferably at least 95%, 96% or more, 97% or more, 98% or more, or 99% or more when the above-mentioned sequences of the present invention are aligned as closely as possible to any other sequence and the aligned sequences are analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are well known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NCBI and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the Internet. BLAST can be accessed at www.ncbi.nlm.nih.gov / BLAST / . Instructions for sequence homology comparison using this program can be found at www.ncbi.nlm.nih.gov / BLAST / blast_help.html.

[0087] Based on this, fusion proteins of the invention may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology to the sequences explicitly set forth herein or to the entirety of the sequences described herein. Such homology may be determined by sequence comparison and / or alignment using methods known in the art. For example, percent sequence identity of nucleic acids or proteins of the invention can be determined using sequence comparison algorithms (e.g., BLAST or BLAST 2.0), manual alignment, or visual inspection.

[0088] The DLL4-binding protein domain is a protein domain that binds to DLL4 and includes the DLL4-binding site of Notch1. For example, it may include the EGF-like domain of human Notch1, and may include the sequence of SEQ ID NO:18.

[0089] The DLL4-binding protein domain may be bound to the terminus of an antibody that binds to VEGFR2 / KDR, for example, the light chain terminus, specifically the light chain N-terminus.

[0090] Korean Patent Registration No. 10-1569083 describes a DIG-KN substance containing VEGFR2 / KDR and DLL4 binding protein domains. However, the present invention comprises an anti-VEGFR2(KDR) antibody in which the heavy chain FR (framework regoin) of a human antibody that specifically binds to VEGFR2 / KDR has been substituted from VH1 to VH3, and the light chain FR (framework regoin) has been substituted from VL1 to VK1, thereby inducing mutations to increase affinity for the antigen. The present invention is different in that a DLL4 binding protein domain (Notch1 EGF-like domain) is bound to the N-terminus of the light chain of the anti-VEGFR2(KDR) antibody.

[0091] The VEGFR2 / KDR-binding antibody or antigen-binding fragment thereof and the DLL4-binding protein domain may be linked via a linker. The linker may be a peptide linker, and may be about 10 -25 It may have any aa length and may include hydrophilic amino acids such as, but not limited to, glycine and / or serine.

[0092] Specifically, the linker may include, for example, (GS)n, (GGS)n, (GSGGS)n, or (GnS)m (n and m are each 1 to 10), but the linker may be, for example, (GnS)m (n and m are each 1 to 10).

[0093] In another aspect, the present invention relates to a nucleic acid encoding the fusion protein.

[0094] The nucleic acid may comprise the sequence of SEQ ID NO: 7, which encodes the heavy chain variable region. The nucleic acid may comprise the sequence of SEQ ID NO: 9, which encodes the light chain variable region. The nucleic acid may comprise the sequence of SEQ ID NO: 17, which encodes the DLL4 binding protein domain.

[0095] Nucleic acids encoding the fusion proteins of the present invention can be isolated and recombinantly produced. The isolated nucleic acids can be inserted into replicable vectors for further cloning (DNA amplification) or further expression. Accordingly, the present invention relates in a further aspect to vectors containing the nucleic acids.

[0096] The term "nucleic acid" encompasses DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acids, include natural nucleotides as well as analogs in which the sugar or base moiety is modified. The sequence of a nucleic acid encoding a fusion protein of the present invention may be modified. Such modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0097] The DNA is readily isolated or synthesized using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to DNA encoding the antibody heavy and light chains). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0098] The term "vector" as used herein refers to a means for expressing a gene of interest in a host cell, and includes plasmid vectors, cosmid vectors, viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors, etc. In the vector, the nucleic acid encoding the fusion protein is operably linked to a promoter.

[0099] "Operably linked" refers to the functional association of a nucleic acid expression control sequence (e.g., a promoter, signal sequence, or an array of transcriptional regulator binding sites) with another nucleic acid sequence, whereby the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0100] When a prokaryotic cell is used as the host, it generally contains a strong promoter capable of driving transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for the initiation of translation, and a transcription / translation termination sequence. Furthermore, for example, when a eukaryotic cell is used as the host, promoters derived from the genome of a mammalian cell (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) may be used, and generally have a polyadenylation sequence as a transcription termination sequence.

[0101] In some cases, the vector may be fused with other sequences to facilitate purification of the fusion protein expressed from the vector, such as glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA).

[0102] The vectors contain antibiotic resistance genes commonly used in the art as selection markers, such as genes for resistance to ampicillin, gentamicin, cabenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0103] In yet another aspect, the present invention relates to a cell transformed with the above-mentioned vector. The cells used to produce the fusion protein of the present invention may be, but are not limited to, prokaryotic, yeast, or higher eukaryotic cells.

[0104] Prokaryotic host cells such as Escherichia coli, strains of Bacillus such as Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus (e.g., Staphylococcus carnosus) can be used.

[0105] However, of greatest interest are animal cells, and examples of useful host cell lines may be, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0106] In yet another aspect, the present invention relates to a method for producing the fusion protein, comprising: (a) culturing the cells; and (b) recovering the fusion protein from the cultured cells.

[0107] The cells can be cultured in a variety of media. Any commercially available medium can be used as the culture medium. Any other necessary supplements known to those skilled in the art may be included at appropriate concentrations. The culture conditions, such as temperature, pH, etc., will be used with the host cell selected for expression and will be apparent to those skilled in the art.

[0108] The fusion protein can be recovered by removing impurities, for example, by centrifugation or ultrafiltration, and the resulting product can be purified using, for example, affinity chromatography. Additional purification techniques, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, and hydroxylapatite chromatography, may also be used.

[0109] The antibody contained in the fusion protein may be an IgG or a fragment containing the variable region, i.e., ScFv or Fab, and the heavy chain variable region may be IgG1, IgG2, IgG3, or IgG4.

[0110] In yet another aspect, the present invention relates to a composition for preventing or treating angiogenic diseases, which comprises the fusion protein as an active ingredient.

[0111] The term "angiogenesis" refers to the formation or growth of new blood vessels from pre-existing blood vessels, and "angiogenesis-related disease" refers to a disease associated with the development or progression of angiogenesis. Any disease that can be treated with the fusion protein may be included in the scope of angiogenesis-related disease without limitation.Examples of angiogenesis-related diseases include cancer, metastasis, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, macular degeneration, neovascular glaucoma, erythrosis, proliferative retinopathy, psoriasis, hemophilic arthritis, capillary formation in atherosclerotic plaques, keloids, wound granulation, vascular adhesion, rheumatoid arthritis, osteoarthritis, autoimmune diseases, Crohn's disease, and the like. Disease, restenosis, atherosclerosis, intestinal adhesions, cat scratch disease, ulcer, liver cirrhosis, nephritis, diabetic nephropathy, diabetic foot ulcers, chronic kidney failure, diabetes mellitus, inflammatory diseases, idiopathic pulmonary fibrosis, sepsis, acute respiratory distress syndrome, and neurodegenerative diseases.

[0112] The cancers include esophageal cancer, stomach cancer, large intestine cancer, rectal cancer, oral cancer, pharynx cancer, larynx cancer, lung cancer, colon cancer, breast cancer, uterine cervical cancer, and endometrial cancer. cancer, ovarian cancer, prostate cancer, testis cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer cancer), leukemia, Hodgkin's lymphoma The cancer is selected from the group consisting of, but not limited to, lymphoma, lymphoma, and multiple myeloid blood cancer.

[0113] As used herein, the term "prevention" or "prophylaxis" refers to any measure of administering a fusion protein of the invention to prevent or delay the onset of a disease of interest. The term "treatment" or "therapy" refers to any measure of administering a fusion protein of the invention to improve or reverse the symptoms of a disease of interest.

[0114] The present invention may be, for example, a pharmaceutical composition for preventing or treating tumors or cancer, comprising (a) a pharmaceutically effective amount of the fusion protein of the present invention; and (b) a pharmaceutically acceptable carrier. The present invention may also be a method for preventing or treating tumors or cancer, comprising administering the fusion protein to a patient with tumor or cancer. The present invention may further be a use of the fusion protein to interfere with a mechanism of action and thereby prevent or treat tumors or cancer.

[0115] Non-limiting examples of tumors or cancers suitable for treatment include melanoma (e.g., metastatic malignant melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate carcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, leukemia, lymphoma, and other neoplastic carcinomas. Additionally, the present invention includes refractory or recurrent cancers that can be treated using the fusion proteins.

[0116] For example, the present invention provides a method for treating an angiogenic disease, comprising the step of administering a therapeutically effective amount of the fusion protein to a patient in need of angiogenesis inhibition. The treatment of an angiogenic disease may further comprise the step of identifying a patient in need of angiogenesis inhibition prior to the administering step. In yet another example, a method for preventing and / or treating an angiogenic disease is provided, comprising the step of administering a therapeutically effective amount of the fusion protein to a patient. The prevention and / or treatment method may further comprise the step of identifying a patient in need of angiogenesis prevention and / or treatment prior to the administering step.

[0117] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, which may be one or more of those commonly used in drug formulations, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. The pharmaceutical composition may also comprise one or more of those commonly used in the manufacture of pharmaceutical compositions, including diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives.

[0118] The pharmaceutical composition or an effective amount of the angiogenic disease can be administered orally or parenterally. Parenteral administration can be by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, or intrarectal administration. Since proteins or peptides are digested during oral administration, oral compositions may be formulated to protect the active agent from degradation in the stomach, for example by coating the active agent. The composition may also be administered by any device capable of transporting the active agent to target cells.

[0119] The compositions of the present invention are administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to a sufficient amount of a pharmaceutical composition for treating a disease at a reasonable benefit / risk ratio applicable to all medical treatments. The effective amount will vary depending on various factors, including the severity of the disease to be treated, the patient's age and sex, the type of disease, the activity of the drug, sensitivity to the drug, the time of administration, the route of administration, the excretion rate, the duration of treatment, co-administration of other drugs, and other parameters well known in the art. The compositions of the present invention may be administered alone or in combination with other therapies. In such cases, they may be administered sequentially or simultaneously with conventional therapies. The compositions may also be administered in a single dose or in multiple divided doses. Taking these factors into consideration, it is important to administer the minimum amount sufficient to obtain maximum efficacy without side effects, and this dosage can be easily determined by experts in the field. The dosage of the pharmaceutical compositions of the present invention is not particularly limited, but will vary depending on various factors, including the patient's health condition and weight, the severity of the disease, the type of drug, the route and time of administration. The compositions may be administered in a single dose or multiple doses daily to mammals, including rats, mice, livestock, humans, etc., by any typically accepted route, for example, orally, rectally, intravenously, subcutaneously, intrauterinely, or intracerebrovascularly.

[0120] The content of the fusion protein in the pharmaceutical composition may vary depending on factors such as formulation method, administration method, patient age, weight, sex, pathological condition, diet, administration time, administration interval, administration route, excretion rate, and reaction sensitivity. For example, the daily dose of the fusion protein may be in the range of 0.001 to 1000 mg / kg, specifically 0.01 to 100 mg / kg, more specifically 0.1 to 50 mg / kg, and even more specifically 0.1 to 20 mg / kg, but is not limited thereto. The daily dose may be formulated as a single formulation in the form of a unit dose, or may be appropriately divided and formulated, or may be packaged in a multi-dose container.

[0121] The pharmaceutical composition can be administered in combination with other drugs, such as other therapeutic agents for angiogenesis inhibition-related diseases, and the dosage, administration method, and type of other drugs may be appropriately prescribed depending on the condition of the patient.

[0122] The pharmaceutical composition may be formulated in the form of a solution, suspension, syrup or emulsion in an oily or aqueous medium, or in the form of an extract, powder, granule, tablet or capsule, etc., and may further contain a dispersing agent or stabilizer for formulation.

[0123] In particular, pharmaceutical compositions containing the fusion protein can be formulated as immunoliposomes because they contain the fusion protein. Liposomes containing the fusion protein can be prepared by methods well known in the art. The immunoliposomes are lipid compositions containing phosphatidylcholine, cholesterol, and polyethylene glycol-derivatized phosphatidylethanolamine, and can be prepared by reverse phase evaporation (Korean Patent Publication No. 10-2015-0089329). For example, Fab' fragments of antibodies can be conjugated to liposomes by disulfide exchange reaction.

[0124] The fusion protein of the present invention may also be used in combination with other pharmaceutical preparations, antibodies, biologically active agents, or substances for various purposes. In this respect, the present invention relates to a composition for combined administration with other therapeutic agents for angiogenic diseases, comprising the fusion protein.

[0125] The other therapeutic agent for angiogenic diseases may include an anti-angiogenic drug, an anti-inflammatory drug, and / or an anti-cancer drug, thereby overcoming resistance to each other and enhancing efficacy.

[0126] When the composition of the present invention is administered in combination with another therapeutic agent for angiogenesis diseases, the fusion protein and the other therapeutic agent for angiogenesis diseases may be administered sequentially or simultaneously. For example, an anti-angiogenic drug, an anti-inflammatory drug, and / or an anti-cancer drug may be administered to a subject, and then a composition containing the fusion protein as an active ingredient may be administered to the subject, or the composition may be administered to a subject, and then the anti-angiogenic drug, the anti-inflammatory drug, and / or the anti-cancer drug may be administered to the subject. Optionally, the composition may be administered to a subject simultaneously with the anti-angiogenic drug, the anti-inflammatory drug, and / or the anti-cancer drug.

[0127]

[0128] Example

[0129] The present invention will be described in more detail below with reference to examples. It will be apparent to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and are not to be construed as limiting the scope of the present invention.

[0130]

[0131] Example 1. CDR grafting

[0132] The CDR sequence of 6A6 was confirmed using Kabat numbering and the IMGT program, and then cloned into human germline VH3 / VK1. The heavy chain CDR-grafted sequence is shown in Figure 1, and the light chain CDR-grafted sequence is shown in Figure 2.

[0133]

[0134] Example 2. Mutation generation and selection for affinity enhancement

[0135] To improve the affinity of the CDR-grafted antibody, we performed optimization. Using soft-randomization, which randomizes the original DNA sequences of the light and heavy chain CDR3s while preserving 70% of the original DNA sequences, we created primers that introduced random mutations into the light chain CDR3 and heavy chain CDR3. We then used these primers to isolate DNA fragments encoding the mutated light chain and heavy chain variable regions through PCR. These DNA fragments were then substituted with the light chain variable region and heavy chain variable region of scFv phage and phagemid, respectively, to create a light chain CDR3 mutant scFv phage library and a heavy chain CDR3 mutant scFv phage DNA library.

[0136] The mutant scFv phage DNA library was purified with phenol-chloroform and then transformed into the Escherichia coli strain XL-1 Blue using electroporation. After confirming the diversity through transformation efficiency analysis and DNA sequence analysis, the cells were cultured at a 500 ml scale to induce phage expression, and a light and heavy chain CDR3 mutant scFv phage library was prepared using the PEG precipitation method.

[0137] Biopanning was performed using each mutant scFv phage library. 100 μl of antigen (KDR1-3 domian-Fc) at 2 μg / ml was added to each well of a 96-well immunoplate and left overnight at 4°C. The next day, the antigen-coated plate was washed three times with PBST (0.1% Tween 20) and then 200 μl of 2% BSA blocking buffer was added and incubated at room temperature for 2 hours. 50 μl of XL1-Blue stock was added to 2 ml of 2x YT-TET (tetracycline 10 μg / ml) growth medium and incubated at 37°C and 200 rpm for approximately 2 hours. 13 ml of XL1-Blue stock was then added and incubated at OD . 600 The cells were grown until the OD reached 0.5. After 2 hours of blocking, the wells were washed three times with 1X PBST (0.1% Tween 20). The mutant library was mixed with the same volume of 4% BSA and then 200 μl was added to each washed well. The wells were then rocked at room temperature for 30 minutes, followed by a 2-hour reaction. After the phage library reaction was complete, the supernatant was discarded and the wells were washed five times with 0.1% PBST and five times with PBS. 100 μl of 100 mM TEA (trimethylamine) was added to each well and shaken at room temperature for 10 minutes. After 10 minutes, 50 μl of 1M Tris (pH 7.5) was added to each well and mixed. The supernatant was analyzed by OD 600The resulting solution was added to 10 ml of XL1-blue with a pH of 0.5 and allowed to infect for 30 minutes at 37°C. After infection, 100 μl was used as the output titer, and the remaining solution was centrifuged at 6,000 rpm for 10 minutes. The supernatant was discarded, and the pellet was spread onto a large square plate (CM 34 μg / ml + 1% glucose) and incubated overnight at 37°C. The remaining 100 μl for the output titer was diluted 1 / 10, 1 / 100, and 1 / 1000, spread onto CM plates, and incubated overnight at 37°C. The next day, the colonies grown on the square plate were added to 50 ml of 2x YT medium, scraped using a loop, and centrifuged at 6000 rpm for 10 minutes. The supernatant was discarded, and the precipitate was used to make a first panning stock. 100 ml of 2x YT culture medium (growth media: CM34 μg / ml + 1% glucose) was placed in a 500 ml Erlenmeyer flask, and the OD 600 Cells were added so that the OD was 0.2, and the cells were incubated at 200 rpm and 37°C. 600 The OD was increased to 0.5. 600After culturing the cells until the RI value reached 0.5, helper phage (M13KO7 mutant) was added at a 20x cell volume. The helper phage was added and the cells were infected at 37°C for 30 minutes, followed by centrifugation at 6000 rpm for 10 minutes. The supernatant was discarded, and the cells were replaced with 100 ml of 2xYT medium (CM 34 μg / ml + Kan 70 μg / ml + 1 mM IPTG + 5 mM MgCl2) and incubated overnight at 200 rpm and 30°C. The next day, the grown cells were centrifuged at 7000 rpm for 10 minutes and then centrifuged once more in the same manner. The collected supernatant was added to 1 / 5 (v / v) of the supernatant in 20% PEG / 2.5 mM NaCl and precipitated on ice for 1 hour. After precipitation, the cells were centrifuged at 9000 rpm for 1 hour. The supernatant was discarded, and the precipitate was dissolved in 5 ml of PBS. It was then filtered through a 0.45 μm filter and stored at 4°C for use in the next panning step. This process was repeated 3-4 times, and antibodies that bound to the antigen were confirmed by ELISA.

[0138] Subsequently, during the selection process, the dissociation rate constant Kdis of the scFv was measured as a quantitative indicator of the ability to retain binding.

[0139]

[0140] Example 3. Selection of antibodies with high affinity to antigen (off-rate screening)

[0141] The antigen binding avidity of the selected antibodies was measured using Octet (Fortebio). To do this, KDR domains 1-3 were immobilized on a biosensor, and then candidate antibodies expressed in the form of scFv were added and allowed to bind, after which the dissociation rate constants were measured. The dissociation rate constants for the five selected optimized clones are shown in Table 1 and their amino acid sequences in Tables 2 and 3.

[0142] [Table 1] Dissociation rate constants of antibodies that specifically bind to antigens JPEG0007796886000001.jpg50128

[0143]

[0144] Example 4. Fusion protein production

[0145] Among the scFv phage antibodies selected for fusion protein production, D4, which had the lowest dissociation constant, was converted to an IgG form. This conversion was performed using molecular biology techniques. Phagemids were extracted from the selected E. coli clones, and the variable regions were amplified using PCR. The amplified heavy chain variable region was inserted into an expression vector containing the heavy chain constant region (Invivogen, pfusess-hchg1), and the amplified light chain variable region was inserted into an expression vector containing the light chain constant region (Invivogen, pfuse2ss-hclk). DNA cloning into the IgG form was completed, and the antibody was converted to an IgG1 form. A DLL4-binding domain (Notch1 EGF-like domain) containing a linker (G4S sequence) was genetically synthesized and fused to the N-terminus of the converted light chain constant region. The nucleotide sequences of the antibodies constructed are shown in Tables 2 and 3.

[0146] [Table 2] CDR sequences of selected antibodies JPEG0007796886000002.jpg36134

[0147] [Table 3] Sequences of selected antibodies JPEG0007796886000003.jpg138135

[0148] JPEG0007796886000004.jpg137139

[0149] JPEG0007796886000005.jpg134136

[0150] JPEG0007796886000006.jpg126128

[0151] JPEG0007796886000007.jpg126129

[0152] JPEG0007796886000008.jpg170128

[0153] JPEG0007796886000009.jpg119128

[0154]

[0155] Example 5. Fusion Protein Expression

[0156] The IgG form was transiently expressed using the Expi293F expression system kit (Thermo Fisher Scientific, US). Expi293 cells included in the kit were cultured in suspension in the dedicated medium at 37°C in a 5% CO2 environment on an orbital shaker at 125 rpm. 3 × 10 cells were cultured every 3 days. 5 The cells were subcultured at a concentration of 3 × 10 cells / ml when the expression vector was introduced. 6 The cell number was adjusted to 1000 cells / ml. The gene transfection was performed using the dedicated reagent Expifectamine. Lipid-DNA complexes containing 1 μg of expression vector DNA and 2.7 μl of Expifectamine per 1 ml of cell suspension were prepared and added to the cell suspension. 16-18 hours after transfection, Enhancer 1 / 2 was added to induce expression. After culturing for 3-4 days under the same conditions, the cells were centrifuged and the IgG-containing supernatant was collected.

[0157]

[0158] Example 6. Purification of fusion proteins

[0159] The resulting supernatant was loaded onto a Protein A column (GE Healthcare) and IgG purified by affinity chromatography. The column was equilibrated with 20 mM Tris-HCl, 50 mM NaCl, and 5 mM EDTA (pH 7.0), after which the supernatant was loaded onto the column and washed with 50 mM Tris-HCl, 500 mM NaCl, 5 mM EDTA, and 0.2% polysorbate 20 (pH 7.0). The column was then eluted with 50 mM NaCl and 0.1 M glycine-HCl (pH 3.5), followed by neutralization with 1 M Tris. The eluted protein was then dialyzed with a 10,000 MWCO spectra / por dialysis membrane (Spectrum Labs, US) to exchange the solvent with PBS. The eluted protein was then concentrated to the required concentration using a Vivaspin (Satorius, DE), aliquoted, and stored at -80°C.

[0160]

[0161] Example 7. Binding specificity analysis of fusion proteins

[0162] The binding affinity of the selected fusion proteins to the antigen was measured using SPR (Biacore T200, Cytiva). The fusion proteins were captured and analyzed using the antigen protein as the analyte. The fusion proteins were immobilized on the surface of a Protein A chip according to the manufacturer's instructions. The fusion proteins were diluted in PBS-T running buffer at a concentration of 5 μg / ml and injected onto the sensor chip at a flow rate of 10 μl / min to reach approximately 200 RU. Antigens were diluted in PBS-T running buffer at different concentrations and injected at a flow rate of 30 μl / min for 4 minutes, with a dissociation time of 10 minutes. To remove bound fusion proteins after each cycle, regeneration solution (10 mM glycine-HCl, pH 1.5) was injected for 1 minute at a flow rate of 30 μl / min. The experimental results were calculated using a 1:1 binding model in BIA evaluation software version 1.0. Table 4 shows the binding strength, and Figures 3A and 3B show the SPR sensorgrams.

[0163] [Table 4] Binding strength of fusion proteins that specifically bind to antigens JPEG0007796886000010.jpg46146

[0164]

[0165] Example 8. Confirmation of simultaneous binding ability of fusion proteins to antigens

[0166] The ability of the selected fusion proteins to simultaneously bind to two antigens was confirmed using SPR (Biacore T200, Cytiva). The fusion proteins were immobilized on the surface of a Protein A chip according to the manufacturer's instructions. The fusion proteins were diluted in PBS-T running buffer at a concentration of 5 μg / ml onto the sensor chip, reaching approximately 200 RU. First, 100 nM hKDR-ECD diluted in PBS-T running buffer was injected at a flow rate of 30 μl / min and allowed to bind for 120 seconds, followed by 60 seconds of running in PBS-T running buffer at a flow rate of 30 μl / min. Without regeneration, 200 nM hDLL4 diluted in PBS-T running buffer was injected at a flow rate of 30 μl / min and allowed to bind for 120 seconds, followed by 60 seconds of running in PBS-T running buffer at a flow rate of 30 μl / min. The results of simultaneous binding of both antigens are shown in FIG.

[0167]

[0168] Example 9. Confirmation of the proliferation ability of fusion proteins in HUVECs

[0169] To compare the effects of the fusion proteins on the proliferation of human umbilical vein endothelial cells (HUVECs), human umbilical vein endothelial cells (HUVECs) were treated with the fusion proteins and control antibodies (6A6, 6A6xDLL4, D4, and Minod-Fc). 6A6xDLL4 is listed in Table 5. Minod-Fc is a fusion protein of Notch1 (SEQ ID NO: 18) and Fc.

[0170] [Table 5] JPEG0007796886000011.jpg46144

[0171] Briefly, HUVEC (LONZA, Cat No. C2519A) cells were cultured on 2% gelatin-coated plates using VascuLife medium complete kit (Lifeline cell technologies, Cat No. LCT-LL-0005). The medium was incubated at 37°C and 5% CO2. HUVECs used for the assay were within passage 10 and were cultured at 1x10 cells per well in a 96-well plate in M199 medium (Gibco, Cat No. 11043-023) containing 0.5% heat-inactivated FBS. 4 Cells were seeded at 1000 μg / well. Each antibody was diluted to 250 nM, 50 nM, 10 nM, or 2 nM and applied to the corresponding wells of an assay plate. The wells were then incubated for 30 minutes. VEGF165 (R&D Systems, Cat. No. 293-VE) was then added at 100 ng / mL per well. A negative control group (VEGF- / Ab-) lacking VEGF or any other drug treatment, and a positive control group (VEGF+ / Ab-) treated with VEGF alone, were incubated for 3 days in a 37°C CO2 incubator. Viability was measured using the CellTier Glo-Luminescent Cell Viability Assay kit (Promega, Cat. No. G7571) to detect ATP in live cells.

[0172] The test results showed that the optimized antibody D4 comparison group and the D4xDLL4 test group, which has a D4 backbone, inhibited VEGF165-mediated HUVEC proliferation to a greater extent than the 6A6 comparison group and the 6A6xDLL4 comparison group, both in a concentration-dependent manner. The results are shown in Figure 5.

[0173]

[0174] Example 10. Inhibitory effect of fusion protein on VEGFR-2 phosphorylation

[0175] To confirm the ability of the fusion proteins to dephosphorylate KDR (VEGFR-2), the fusion proteins and control antibodies (6A6, 6A6xDLL4, D4, Minod-Fc) were compared by Western blot analysis.

[0176] Briefly, HUVEC (LONZA, Cat No. C2519A) cells were cultured in a 6-well plate at 8 x 10 cells per well using VascuLife medium complete kit (Lifeline cell technology, Cat No. #LCT-LL-0005). 5Cells were seeded at a concentration of 1000 cells / well and cultured at 37°C and 5% CO2 for one day. HUVEC cells were treated with each antibody at 125 nM or 25 nM for 30 minutes, followed by VEGF165 (R&D, Cat No. 293-VE) at 50 ng / mL per well for 10 minutes. A negative control group (VEGF- / Ab-) was treated with neither VEGF nor drug, and a positive control group (VEGF+ / Ab-) was treated with VEGF alone. Drug-treated HUVECs were washed with phosphate-buffered saline and then lysed in lysis buffer (Thermo Scientific, RIPA buffer and phosphatase inhibitor) on ice for 10 minutes. Lysed cells were collected using a cell scraper and centrifuged at 13,000 rpm at 4°C for 10 minutes to obtain the supernatant. The total protein content of the resulting supernatant was quantified using the BCA protein assay. 20 μg of each sample was loaded onto a 4-15% grade SDS-PAGE gel and subjected to electrophoresis. After electrophoresis, the gel was transferred to a nitrocellulose membrane (Bio-Rad, Trans-Blot Turbo Transfer System) for Western blot analysis. The nitrocellulose membrane blot was blocked with blocking buffer [5% skim milk / TBS-T (0.1% Tween 20, Tris-based saline)] at room temperature for 1 hour. After washing with TBS-T buffer, the primary antibody (p-VERFR2 (Y1175), Cell Signaling) was diluted 1:1000 and incubated at room temperature for 1 hour. After washing, the secondary antibody (anti-rabbit-HRP) was diluted 1:5000 and incubated at room temperature for 1 hour.After washing with TBS-T buffer, the cells were treated with ECL (Enhanced chemiluminescence) solution (Thermo Fisher Scientific, Cat No. 34096) and reacted, after which images were obtained using a Luminescent Image Analyzer (GE Healthcare, Amersham Imager 680).

[0177] The test results showed that VEGF165-mediated VEGFR2 phosphorylation was partially inhibited in a concentration-dependent manner in the 6A6 control group and the 6A6xDLL4 control group, whereas VEGFR2 was almost completely dephosphorylated in the test groups treated with the same amount of the optimized antibody, D4 control group and D4xDLL4. These results are shown in Figure 6.

[0178]

[0179] Example 11. Confirmation of KDR (VEGFR-2) binding ability using FACS

[0180] To examine the binding activity of the fusion proteins to HUVECs, FACS was performed using the fusion proteins and control antibodies (6A6, 6A6xDLL4, D4, Minod-Fc).

[0181] Briefly, HUVEC (LONZA, Cat No. C2519A) cells were cultured in VascuLife medium complete kit (Lifeline cell technology, Cat No. LCT-LL-0005) medium on 2% gelatin-coated plates at 37°C and 5% CO. Cells were cultured in FACS buffer (0.5% FBS, 0.05% sodium azide in PBS) at a density of 2 × 10 7The cells were prepared at a concentration of 100 μL per FACS tube and 50 μL of each antibody was dispensed into FACS tubes. 100 μL of each antibody at 5 nM concentration was mixed into each FACS tube and incubated at 4°C for 30 minutes. After the incubation, 2 mL of FACS buffer was added to each tube and centrifuged at 1500 rpm for 3 minutes. The supernatant was removed, and the cells were resuspended. 100 μL of anti-human IgG-PE antibody (1:200, Bethyl, #A80-248PE) was added to each tube and incubated at 4°C for 20 minutes. 2 mL of FACS buffer was added to each FACS tube and centrifuged at 1500 rpm for 3 minutes. The separated supernatant was removed, and the cells were resuspended in 200 μL of FACS buffer. Binding activity was analyzed using a FACS (Beckton Dickinson, Lyric).

[0182] The results of the study confirmed that, except for Minod-Fc, all of the VEGFR2-targeting antibodies 6A6, 6A6xDLL4, D4, and D4xDLL4 bound to HUVECs. Furthermore, D4 ​​and D4xDLL4 showed improved binding activity compared to 6A6 and 6A6xDLL4. These results are shown in Figure 7.

[0183]

[0184] Example 12. Confirmation of antibody binding ability to hDLL4 expressed on the cell surface

[0185] To generate cells expressing DLL4 on the cell surface, HEK293 cells were transformed with pcDNA-hDLL4.

[0186] Briefly, the pcDNA-hDLL4 vector synthesized at GeneArt was transfected into HEK293 cells using lipofectamine. TMThe cells were transfected using 2000 (Invitrogen #1168-019, USA) and selected by adding neomycin (Gibco #10131, 50 mg / mL). All growing cells were then pooled and cultured. Then, 23 single colonies were obtained by culture using the limiting dilution method. One cell line that highly overexpressed hDLL4 was selected for the experiment. The selected cells were placed in FACS buffer (0.5% FBS, 0.05% sodium azide in PBS) at 2 x 10 7 The cells were prepared at a concentration of 100 μL / mL and dispensed into FACS tubes at a volume of 50 μL each. 100 μL of 5 nM fusion protein and control antibodies (6A6, 6A6xDLL4, D4, D4xDLL4, Minod-Fc) were added to the FACS tubes containing the cells and incubated at 4°C for 30 minutes. After the incubation, 2 mL of FACS buffer was added to each tube and the tubes were centrifuged at 1500 rpm for 3 minutes. The supernatant was removed, the cells were thoroughly suspended, and 100 μL of anti-human IgG-PE antibody (1:200, BioLegend, #A80-248PE) was added to each tube and incubated at 4°C for 20 minutes. After the incubation, 2 mL of FACS buffer was added to each FACS tube and the tubes were centrifuged at 1500 rpm for 3 minutes. The separated supernatant was removed, and the cells were suspended in 200 μl of FACS buffer each, followed by analysis of binding activity using FACS (Beckton Dickinson, Lyric).

[0187] The results showed that only Minod-Fc, 6A6xDLL4, and D4xDLL4, which contain the Minod domain that targets DLL4, bound to HEK293 cells overexpressing DLL4 (DLL4-overexpressing HEK293), and that the fusion proteins 6A6xDLL4 and D4xDLL4 bound better than Minod-Fc alone. 6A6 and D4 did not bind to HEK293 cells overexpressing DLL4. These results are shown in Figure 8.

[0188] [Industrial Applicability]

[0189] The present invention provides a fusion protein that simultaneously targets VEGFR2 / KDR and DLL4, and contains an antibody with improved affinity compared to existing antibodies targeting VEGFR2 / KDR, thereby providing more effective combined therapy and monotherapy in tumor treatment than existing therapeutic agents. The present invention exhibits improved binding to VEGFR2 / KDR, making it useful for preventing or treating targeted cancers / tumors or diseases associated with angiogenesis inhibition. By simultaneously inhibiting Notch1-mediated DLL4 activity, the present invention provides superior angiogenesis inhibition ability compared to single treatments.

[0190]

[0191] Although certain parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and limit the scope of the present invention. Therefore, the true scope of the present invention is to be defined by the appended claims and their equivalents.

[0192] Sequence Catalog Free Text

[0193] Attach as an electronic file

Claims

1. A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR; and a DLL4 (Delta-like ligand 4) binding protein domain, The antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR is a fusion protein comprising a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, and a heavy chain CDR3 of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO:

6.

2. The fusion protein of claim 1 , wherein the antibody that binds to VEGFR2 / KDR comprises a heavy chain variable region of SEQ ID NO: 8 and a light chain variable region of SEQ ID NO:

10.

3. The fusion protein of claim 1 , wherein the DLL4 binding protein is Notch1 comprising the sequence of SEQ ID NO:

18.

4. The fusion protein of claim 1 , wherein the DLL4 binding protein domain binds to the N-terminus of the light chain of an antibody that binds to VEGFR2 / KDR.

5. The fusion protein according to claim 1, wherein the antibody or antigen-binding fragment thereof that binds to VEGFR2 / KDR and the DLL4 binding protein domain are linked via a linker.

6. The linker may be ｎ S) ｍ (n, m are each 1 to 10).

7. A nucleic acid encoding the fusion protein according to any one of claims 1 to 6.

8. An expression vector comprising the nucleic acid of claim 7.

9. A transformed cell comprising the expression vector of claim 8.

10. A method for producing a fusion protein comprising the steps of: (a) culturing the cell of claim 9 to produce the fusion protein; and (b) recovering the produced fusion protein.

11. A composition for preventing or treating angiogenic diseases, comprising the fusion protein according to any one of claims 1 to 6 as an active ingredient.

12. A composition for diagnosing angiogenic diseases, comprising the fusion protein according to any one of claims 1 to 6 as an active ingredient.

13. A composition for preventing or treating tumors or cancers, comprising the fusion protein according to any one of claims 1 to 6 as an active ingredient.

14. A composition for combined administration with other therapeutic agents, comprising the fusion protein according to any one of claims 1 to 6.

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